Mass spectrometry has become the method of choice for fast and efficient identification of proteins in biological samples. In particular, tandem mass spectrometry of peptides derived from a complex protein mixture can be used to identify and quantify the proteins present in the original mixture. In general practice, such information is obtained by ideally selecting and isolating single ion species (of a single mass-to-charge ratio, or m/z, value or of a restricted range of m/z values) and subjecting such so-isolated precursor ions to fragmentation so as to yield product ions that can be used to identify peptides. Ion fragmentation can be provided by various methodologies and mechanisms including collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD). In these dissociation methods, kinetic or electromagnetic energy is imparted to the peptide ions, whereby the introduced energy is converted into internal vibrational energy that is then distributed throughout the bonds of the peptide ions. When the energy imparted to a particular bond exceeds that required to break the bond, fragmentation occurs and product ions are formed. Other mechanisms of fragmentation include for example, those in which the capture of a thermal electron is exothermic and causes the peptide backbone to fragment by a non-ergodic process, those that do not involve intramolecular vibrational energy redistribution. Such methodologies include Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD). ECD and ETD occur on a time scale that is short compared with the internal energy distribution that occurs in the CID process, and consequently, most sequence specific fragment forming bond dissociations are typically randomly along the peptide backbone, and not of the side-chains.
The information that is derived from tandem mass spectrometry experiments comprises a list of m/z values of fragment ions as well as correlations between the fragment-ion m/z values and the m/z values of the precursor ions from which the fragments were derived. This information can be used to search peptide sequence databases to identify the amino acid sequences represented by the spectrum and, thus, to identify the protein or proteins from which the peptides were derived. To identify peptides, database searching programs typically compare each MS/MS spectrum against amino acid sequences in the database, and a probability score is assigned to rank the most likely peptide match.
Because tandem mass spectra of peptide mixtures are generally complex, data-dependent data acquisition techniques have been developed in order to systemize mass spectral analyses. During data-dependent acquisition, an initial survey mass spectrum of potential precursor ions is obtained prior to fragmentation. Automated processing of the survey mass spectrum identifies the most abundant ionized species which are then selected for subsequent isolation and fragmentation followed by mass analysis of fragments (Fejes et al. Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris. Photosynth Res. 2003; 78(3):195-203). If data is being obtained from a sample undergoing chromatographic separation, this sequence of events may be repeated as each fraction elutes (i.e., at each of a plurality of chromatographic retention times). A data-dependent method that makes use of this process is schematically illustrated at 10 in FIG. 1A.
Boxes 12 in FIG. 1A schematically represent survey mass spectra that are conducted so as to determine the m/z values of the various ion species that are introduced into a mass spectrometer at any particular time. Since mass-to-charge ratio (m/z) values are represented as ordinate values and chromatographic retention time values are represented as abscissa values in FIG. 1A, the height of the boxes 12 represents the m/z range of the survey mass spectral measurements. For purposes of example only, the common height of the boxes 12 is representing a survey mass spectral range from 400 Da to 1200 Da, which is a common range of interest. Each survey mass spectrum (or, equivalently, survey “scan”) is a measurement of the relative abundances and mass-to-charge ratios of first-generation ion species as produced by an ion source and as delivered to a mass analyzer and possibly including some proportion of fragment ions generated in an uncontrolled fashion by in-source fragmentation. The positions of the boxes 12 represent the various times (retention times) at which the survey mass spectra are obtained and correspond to the elution of different respective sample fractions that are introduced into a mass spectrometer. Generally, such survey spectra will be obtained at approximately regular time intervals. Although only five survey mass spectra are indicated in FIG. 1A, the actual number of such survey spectra obtained during the course of an LCMS experiment may hundreds or even thousands. The widths of the boxes shown in FIG. 1A do not have any significance; generally, the time required to obtain any individual mass spectrum is exceedingly small relative to the time over which elution occurs.
According to a so-called “shotgun” type of data-dependent analysis, each survey mass spectrum is automatically analyzed, in real-time during the course of the experiment, to identify the most abundant first-generation ions being introduced into the mass spectrometer at the time of the survey measurement. The most abundant ions give rise to the most intense lines in the mass spectrum. Thus, the m/z values of the most intense lines are identified and recorded. Subsequently, an ion species having each identified m/z value (more correctly, having a restricted, isolated range of m/z that encompass a particular identified m/z value) is respectively isolated within the mass spectrometer and subjected to fragmentation in a collision cell or other fragmentation cell so as to generate one or more fragment ions (product ion species). The isolated first-generation ion species and ions that are to be fragmented or that have been fragmented to produce identified product ion species are herein referred to as “precursor ion species” or “precursor ions”. Each one of the boxes 14 in FIG. 1A schematically represent an occurrence of isolation of a particular ion species followed by fragmentation of that ion species and analysis of the so-generated product ions. The ordinate position of each box 14 represents the m/z value of a hypothetical observed precursor ion; the product ions generated by fragmentation of each precursor ion may comprise a range of product-ion m/z values (not specifically indicated by any box) throughout the measurement range of interest. The occurrence of ten such boxes 14 after the occurrence of each one of the first four survey mass spectra (boxes 12) are shown so as to represent the identification, isolation and fragmentation of each of ten most abundant precursor ion species. The different patterns of boxes 14 after each one of the first four survey mass spectra represents that the signatures of different ion species may dominate different survey spectra, since the appearances of different ion species correlate with the chromatographic elution of different respective compounds.
FIG. 4A illustrates a generalized schematic depiction of an analysis system 200 comprising a liquid chromatograph and mass spectrometer (e.g., an LCMS system) as may be employed to generate tandem mass spectra corresponding to mass spectral experiments of the type discussed in this document. In the system 200, a liquid chromatograph 202 provides a stream of liquid eluate to an ion source 204 of the mass spectrometer through a fluidic conduit 203. The ion source which may, without limitation, comprise an electrospray, thermospray or Atmospheric Pressure Chemical Ionization (APCI) source generates a plume of ions of various species which are introduced into an evacuated chamber 206 of the mass spectrometer through an aperture or tube 207 thereof.
A first set of ion optical components 208a of the mass spectrometer of the analysis system 200 directs the ions into an ion selection, mass analysis or storage device 210 which may comprise, without limitation, a quadrupole mass filter, a quadrupole ion trap or a quadrupole mass analyzer. In some modes of operation, the device 210 may be operated so as to isolate a selected population of ion species, in accordance with a selected m/z value or range of m/z values. In other modes of operation, the device 210 may be operated so as to generate a mass spectrum or mass spectra of the ions that are introduced into the evacuated chamber. A second set of ion optical components 208b directs ions from the device 210 into a fragmentation cell 212. The fragmentation cell may operate according any one of several mechanisms including, without limitation, collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD).
Fragment ions (i.e., product ions) generated within the fragmentation cell 212 are directed, by means of a third set of ion optical components 208c, to a mass analyzer 214 that includes an ion detector 216. The mass analyze 214 may be any one of various different mass analyzer types and may comprise, without limitation, a quadrupole mass filter, a quadrupole ion trap, a time-of-flight (TOF) mass analyzer, a magnetic sector mass analyzer, an electrostatic trapping mass analyzer, such as an orbital trapping mass analyzer or a Cassini trap mass analyzer or a Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass analyzer. Each mass spectrum, which may be of either precursor ion species or product ion species, that is generated by the mass analyzer 214 and detector 216 is a record of relative detected abundances of ions of different m/z values.
The detector 216 of the analysis system 200 (FIG. 4A) communicates such mass spectral data to an electronic controller 218, such as a computer, circuit board, or set of modular integrated circuit components, over an electronic communication line 221. Other electronic communication lines 221 may also be present within the system 200 so as to electronically couple the controller 218 to the chromatograph 202, the ion source 204, the ion selection, mass analysis or storage device 210, the fragmentation cell 212, the mass analyzer 214 or the various ion optical assemblies (208a-208c). The electronic communication lines 221, which may be either unidirectional or bi-directional, may be employed to send operational instructions from the controller to any of these various components (as well as others) or to receive information from any of these components (as well as others). The controller 218 includes computer-readable electronic memory 219 and may operate according to control instructions (such as a computer program) stored on the electronic memory. The control instructions may comprise instructions to cause the various components of the analysis system 200 to operate in a coordinated fashion so as to execute various mass spectrometry methods as described in this document.
Although the system 200 has been described in terms of LCMS as comprising a liquid chromatograph 202 that supplies a chemically fractionated sample to a mass spectrometer, it should be kept in mind that, alternatively, an unfractionated sample could be supplied to the mass spectrometer through simple infusion or that, still further alternatively, some other form of chemical separation technique or chemical fractionation technique could be used in conjunction with or in place of the chromatograph 202. For example, the system could make use of apparatus corresponding to additional or other techniques that are known in the art of chemical separation, such as liquid-liquid extraction, solid phase supported liquid extraction, random access media column extraction, monolithic column extraction, dialysis extraction, dispersive solid phase extraction, solid phase micro-extraction, etc. Such alternatively configured systems may also be employed to generate tandem mass spectra corresponding to mass spectral experiments of the type discussed in this document.
In many instances, certain method steps may be advantageously performed using a mass spectrometer system that comprises more than one mass analyzer. FIG. 4B schematically illustrates one such system, which is marketed and sold under the Thermo Scientific™ Orbitrap Fusion™ mass spectrometer name by Thermo Fisher Scientific of Waltham, Mass. USA. The system 300 illustrated in FIG. 4B is a composite system comprising multiple mass analyzers including: (a) a dual-pressure linear ion trap analyzer 340 and (b) an ORBITRAP™ oribital trapping mass analyzer (a type of electrostatic trap analyzer) 360. A key performance characteristic of this instrument is its high duty cycle, which is realized by efficient scan scheduling, so that survey mass spectra are acquired with one analyzer while product-ion mass spectra are acquired with the other analyzer. In addition to the two mass analyzers, the system 300 further includes a quadrupole mass filter 333 which may be employed for isolation of various ranges of precursor ions, a C-trap ion trap 350 which is operational to route ions into the Orbitrap™ mass analyzer and an ion-routing multipole ion guide 309 which may be configured to either store ions or fragment ions by collision-induced dissociation (CID) and is capable of routing ions in the direction of either the C-trap ion trap 350 or the dual-pressure linear ion trap analyzer 340.
The dual-pressure linear ion trap analyzer 340 comprises a high-pressure cell portion 340a and a low-pressure cell portion 340b. The high-pressure cell portion 340a may be infused with either an inert gas for purposes of enabling ion fragmentation by collision-induced dissociation or with a reagent gas for purposes of enabling ion fragmentation by electron transfer dissociation (ETD). The low-pressure cell portion 340b is maintained under high vacuum and includes ion detectors 341 for operation as a linear ion trap mass analyzer. Thus, the system 300 provides ion fragmentation capability in either the multipole ion guide 309 or in the high-pressure cell portion 340a of the dual-pressure linear ion trap analyzer 340.
In operation of the system 300, ions introduced from ion source 312 are efficiently guided and focused into an evacuated chamber by stacked ring ion guide 302. A bent active beam guide 307 causes ions to change their trajectory whereas neutral molecules follow a straight-line trajectory which enables them to be vented by the vacuum system (not illustrated). The ions then pass into the quadrupole mass filter which may be operated, in known fashion, such that only ions comprising a certain pre-determined m/z range or ranges pass through in the direction of the C-trap 350. From the C-trap, ions may be directed into the ORBITRAP™ oribital trapping mass analyzer for high-accuracy mass analysis or may be caused to pass into the multipole ion guide 309 or the ion trap analyzer 340 for either fragmentation, mass analysis or both. After fragmentation, product ions may be routed back to the C-trap 350 for subsequent injection into the ORBITRAP™ oribital trapping mass analyzer for high-accuracy mass analysis.
FIG. 1B is a schematic illustration of a hypothetical sequence of events and hypothetical investigated m/z ranges in accordance with a conventional targeted mass analysis procedure which may be variously known or referred to as selected ion monitoring (SIM), selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). The targeted analysis method shown generally at 20 of FIG. 1B makes use of the fact that, for many biological molecules, a highly reliable identification may be made by detecting a precursor ion species of a particular m/z value and, subsequently, after fragmenting that ion species, detecting fragment ions of one or more particular product-ion m/z values.
As previously described with regard to FIG. 1A, m/z values of precursor (first-generation) ion species are represented as ordinate values and chromatographic retention time values are represented as abscissa values in FIG. 1B. Survey mass spectra are illustrated by hollow boxes 22a-22d. Boxes 22a represent survey spectra that are conducted so as to detect a first-generation ion species having an m/z value of m1, if present. Likewise, boxes 22b, 22c and 22d represent survey spectra that are conducted so as to detect, if present, different first-generation ion species having m/z values of m2, m3, and m4, respectively. These targeted m/z values (m1-m4) are selected in advance of the experiment. As one example, each such ion species may possibly represent the presence, in the eluate, of a respective particular compound of interest. Because only specific ion species are searched for in a targeted experiment, each survey mass spectrum (22a-22d) is designed to analyze only a relatively narrow m/z range about the targeted value.
Because different compounds chromatographically elute at different times, specifically targeted ions will not be detected at all times. The targeted ion species will only be detected during the elution of the respective corresponding compound of interest (that gives rise to the respective ion species) or during elution of some other compound that gives rise to an ion species that coincidentally comprises an m/z value similar to that of the targeted ion species. Once the targeted m/z value is detected (and only when it is detected), the detected ion species is isolated and fragmented and the resulting fragment (product) ions are mass analyzed. The detection, fragmentation and product-ion investigation of precursor ions having m/z values of m1, m2, m3 and m4 are respectively indicated by lines 24a, 24b, 24c and 24d in FIG. 1B. Accordingly, FIG. 1B indicates that a compound that gives rise to a precursor ion species having an m/z value of m1 elutes approximately between time t2 and time t5, inclusive (range 26a). Likewise, as indicated in the same figure, ion species having the m/z values of m1, m2, m3 and m4 elute within the ranges 26b, 26c and 26d, respectively. Once a compound of interest has been detected, by recognition of one or more targeted precursor-ion m/z values and one or more targeted product-ion m/z values, then these m/z values may be excluded from further searches by placement on a so-called “exclusion list”. Such exclusion is indicated by the dotted-line boxes 22a, 22b and 22c in FIG. 1B. Note that the product ions generated by fragmentation of each precursor ion may comprise a range of m/z values (not specifically indicated by any box) throughout the measurement range of interest.
With regard to most analyses of biological samples, neither of the data-dependent analysis methods indicated at 10 in FIG. 1A or at 20 in FIG. 1B is capable of generating a fully comprehensive list of all proteins or peptides that may be present in a sample. The targeted analysis method (FIG. 1B) is not designed to do so. With regard to the shotgun approach (FIG. 1A), numerous studies showing the non-reproducible nature of peptides detected in replicate analyses of the same sample (Panchaud et al. Faster, quantitative, and accurate precursor acquisition independent from ion count. Anal Chem. 2011 Mar. 15; 83(6):2250-7) have demonstrated that that such methods fail to provide full coverage of peptides in a complex mixture. The shotgun approach only detects the most abundant peptides; numerous other low-abundance peptide compounds that may co-elute together with the abundant peptides remain below a requisite intensity threshold or are indistinguishable from spectral “noise”. Moreover, when numerous peptides co-elute, the nature of the chromatographic experiment does not provide sufficient time for separate isolation, fragmentation and fragment analysis for every possible candidate m/z value.
The analysis technique known as “data-independent acquisition” was developed in an attempt expand the number of proteins and peptides that may be detected by LCMS analysis of natural samples. Such expanded coverage could aid an understanding of the complexity of the proteome and the significance of the low-abundance proteome. Such experiments are generally performed without isolation of specific first-generation ion species as precursor ions. Instead, reliance is placed upon computational mining of comprehensive mass spectral data sets obtained from experiments in which first-generation ion species encompassing a wide range of m/z values are simultaneously fragmented so as to generate complex product-ion spectra containing multiplexed signatures of all fragment ions. Although data-independent acquisition methods can provide a comprehensive list of all possible fragment ions, there is generally no direct recorded “parent-child” relationship between precursor ions and fragment ions. Such methods have been made possible by improvements in mass spectrometer speed, accuracy and resolution (thereby limiting interferences between a multitude of mass spectral lines) as well as by the development of mass spectral libraries and advanced computational processing techniques.
FIG. 1C is a highly schematic diagram, shown generally at 30, illustrating the general sequence of events that may occur during a hypothetical LCMS analysis performed according to one data-independent acquisition method known as “SWATH MS” (Gillet et al., Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis, Mol. Cell. Proteomics, 2012, 11(6):O111.016717. DOI: 10.1074/mcp.O111.016717) and often used in conjunction with SWATH™ quantitative proteomics software. In similarity to previously discussed diagrams, m/z values of precursor ions or first-generation ions are represented as ordinate values and chromatographic retention time values are represented as abscissa values. The SWATH MS data-independent procedure includes consecutively acquiring a series of high-resolution, accurate-mass fragment-ion spectra during an entire chromatographic elution (retention time) range by repeatedly stepping through a number (for example thirty two) discrete precursor-ion isolation windows of a certain width (for example, 25 Da width) across a full mass spectral range of interest (for example, the 400-1200 m/z range). Thus, a main feature of the technique, as illustrated in FIG. 1C, is a plurality of series of consecutive product-ion analyses 34. Each such product ion analysis 34 is represented as a shaded box and includes the steps of: isolation of precursor ions within a restricted range of m/z values, fragmentation of the isolated precursor ions so as to generate fragment ions and mass analysis of the fragment ions generated from the isolated precursor ions (i.e., a fragmentation scan). Each restricted range of precursor m/z values may be termed an “isolation window” (or, equivalently, an “isolation range” or an “isolated range”) and is represented by the range of ordinate values that is spanned by a respective one of the boxes 34. For example, the isolation ranges represented by the first several boxes 34, beginning at the lower left position, of FIG. 1C are 400-430 Da, 420-450 Da, 440-470 Da, 460-490 Da, 480-510 Da, etc. Isolation ranges are indicated similarly in other of the accompanying figures. The width of the isolation windows (height of the boxes 34) is significantly greater than those of isolation windows employed in standard shotgun and targeted (FIGS. 1A-1B) methods and are represented, in FIG. 1C, by the height of the shaded boxes that represent the product ion analyses. It should be noted that the product ions, themselves, that are generated by fragmentation of set of precursor ions may comprise a different range of product-ion ink values (not specifically indicated by any box).
Two series, 35a and 35b, of product-ion analyses are illustrated in FIG. 1C. Consecutive isolation windows (corresponding to consecutive product-ion analyses) partially overlap one another in m/z to assure that there are no ink gaps within which ink positions of unfragmented first-generation ions occur. Once the series of isolation windows has covered the full ink range of interest (i.e, once an end of the full ink range of interest has been reached), then a new series of consecutive product-ion analyses is investigated in similar fashion starting at the opposite end of the range. As used herein, the term “cycle time” is the time required to return to the acquisition of any given precursor isolation window. The boxes 32 outlined with dashed lines at the beginning of each cycle depict optional acquisition of a high-resolution, accurate mass survey scan of precursor ions throughout the full ink range of interest. The totality of data product-ion analyses 34 corresponding to any given precursor mass range across the range of retention times is oftentimes referred to as a “swath”. One such swath is shown at 38 in FIG. 1C.
After the collection of mass spectral data as depicted in FIG. 1C, certain targeted peptide or protein compounds may be recognized by mathematical processing of the data. Conventional peptide database search engines, as utilized in conjunction with the shotgun technique illustrated in FIG. 1A, require information relating to which specific fragment ions (more correctly, which ink values) are generated from any given precursor m/z. Disadvantageously, such information is not generally recorded using the data-independent acquisition method illustrated in FIG. 1C. Therefore, such data-independent acquisition methods cannot use conventional database search engines for data processing. Instead, the targeted data processing used to mine the complex data set generated by a data-independent experiment such as that illustrated in FIG. 1C makes use of reference mass spectral libraries. Such libraries may include previously determined reference spectra of known compounds and may include information such as the m/z positions and relative intensities of mass spectral lines as well as chromatographic retention times and other associated information. To perform the targeted data extraction of information (for example, relating to a peptide of interest) from an experiment of the type illustrated in FIG. 1C, the most intense fragment ions of the peptide of interest are retrieved a reference mass spectral library. Patterns of correlated fragment-ion m/z positions, relative intensities and elution profiles are then matched to the reference information to recognize patterns of signals that can uniquely identify the targeted compound or compounds.
Although data-independent mass spectral acquisition methods similar to that schematically illustrated in FIG. 1C have been successfully employed in various circumstances, they may be associated with various disadvantages in certain other circumstances. For example, when measuring highly complex mass spectra, a potential problem of fragment ion interference depends on the product-ion analysis isolation window width. For instance, a wide window width decreases cycle time, which is advantageous when elution peaks are of short-duration, as is characteristic of good chromatographic separation. However, the same wide window width increases the chance of co-isolation of many first-generation ion species, including interfering background ions, prior to fragmentation, thereby increasing the possibility of interferences in the product ion spectra. Decreasing the window width may be expected to decrease the number of first-generation ion species that are co-isolated but, in this instance, the chromatography must be of poorer resolution in order to accommodate the resulting longer cycle times. Further, the rate of product ion interference also depends on the mass accuracy and resolution of the fragment isolation window during data analysis. There remains a need for improved methods of mass spectral analysis of complex mixtures of biological molecules.